Structural Perspectives on the Mechanism of Soluble Guanylate Cyclase Activation

International Journal of Molecular Sciences

Review Structural Perspectives on the Mechanism of Soluble Guanylate Cyclase Activation

Elizabeth C. Wittenborn and Michael A. Marletta *

California Institute for Quantitative Biosciences, Departments of Chemistry and of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; [email protected] * Correspondence: [email protected]

Abstract: The enzyme soluble guanylate cyclase (sGC) is the prototypical nitric oxide (NO) receptor in humans and other higher eukaryotes and is responsible for transducing the initial NO signal to the secondary messenger cyclic guanosine monophosphate (cGMP). Generation of cGMP in turn leads to diverse physiological effects in the cardiopulmonary, vascular, and neurological systems. Given these important downstream effects, sGC has been biochemically characterized in great detail in the four decades since its discovery. Structures of full-length sGC, however, have proven elusive until very recently. In 2019, advances in single particle cryo–electron microscopy (cryo-EM) enabled visualization of full-length sGC for the ﬁrst time. This review will summarize insights revealed by the structures of sGC in the unactivated and activated states and discuss their implications in the mechanism of sGC activation.

Keywords: nitric oxide; soluble guanylate cyclase; cryo–electron microscopy; enzyme structure

Citation: Wittenborn, E.C.; Marletta, 1. Introduction M.A. Structural Perspectives on the Soluble guanylate cyclase (sGC) is a nitric oxide (NO)-responsive enzyme that serves Mechanism of Soluble Guanylate as a source of the secondary messenger cyclic guanosine monophosphate (cGMP) in Cyclase Activation. Int. J. Mol. Sci. humans and other higher eukaryotes [1]. Upon NO binding to sGC, the rate of cGMP 2021, 22, 5439. https://doi.org/ formation increases by several hundred-fold, effectively amplifying the initial NO signal. 10.3390/ijms22115439 cGMP in turn regulates downstream effectors, such as cGMP-dependent protein kinases (PKGs), ion channels, and phosphodiesterases (PDEs), leading to a variety of physiological Academic Editor: Jens Schlossmann responses that include vasodilation, inhibition of platelet aggregation, and effects on neu- rotransmission [2,3]. As a result, dysregulation of the NO-sGC-cGMP signaling pathway Received: 3 May 2021 is associated with various disease pathologies, including hypertension, cardiovascular Accepted: 18 May 2021 Published: 21 May 2021 diseases, neurodegenerative diseases, and asthma [4–8]. Indeed, stimulators of sGC activity have proven effective in the treatment of pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension. The ﬁrst sGC-targeted drug, Adempas®, was Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in approved by the US Food and Drug Administration in 2013 [9]. Given this clinical relevance, published maps and institutional afﬁl- sGC has received considerable attention over the years, with research efforts aimed at better iations. understanding the complex mechanism of activity regulation. Biochemical aspects of sGC activation and deactivation have been reviewed recently [10] and this review will focus on recent and exciting advances in our knowledge of sGC from a structural perspective.

2. Domain Architecture of sGC Copyright: © 2021 by the authors. α β Licensee MDPI, Basel, Switzerland. sGC is an obligate heterodimer consisting of homologous and subunits that share This article is an open access article roughly 30% sequence identity, depending on the species of origin. Each subunit contains distributed under the terms and four domains: an N-terminal heme NO/O2-binding (H-NOX) domain, followed by a conditions of the Creative Commons Per-Arnt-Sim (PAS) domain, a coiled coil (CC) domain, and a C-terminal catalytic (CAT) Attribution (CC BY) license (https:// domain (Figure1A). As its name implies, the H-NOX domain contains a heme cofactor creativecommons.org/licenses/by/ that binds a single equivalent of NO at the iron center [11–13]. Although both α and β 4.0/). subunits contain an H-NOX domain, only the β H-NOX domain contains a heme [12,14].

Int. J. Mol. Sci. 2021, 22, 5439. https://doi.org/10.3390/ijms22115439 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 18

Arnt-Sim (PAS) domain, a coiled coil (CC) domain, and a C-terminal catalytic (CAT) do- Int. J. Mol. Sci. 2021, 22, 5439 main (Figure 1A). As its name implies, the H-NOX domain contains a heme cofactor2 ofthat 18 binds a single equivalent of NO at the iron center [11–13]. Although both α and β subunits contain an H-NOX domain, only the β H-NOX domain contains a heme [12,14]. PAS do- PASmains domains are widely are widely found foundin signaling in signaling proteins proteins where where they serve they serveas sensing as sensing domains domains or as ormediators as mediators of protein–protein of protein–protein interactions interactions [15]. CC [15 domains]. CC domains are versatile are versatile structural structural motifs motifsthat vary that greatly vary greatlyin both in size both and size function, and function, but arebut commonly are commonly found C-terminal found C-terminal to PAS todomains PAS domains where wherethey are they thought are thought to contribute to contribute to signal to signal transduction transduction between between the PAS the PASdomain domain and andan effector an effector domain domain [15–17]. [15–17 Th]. Thee sGC sGC CAT CAT domain domain is is a a class class III nucleotidenucleotide cyclasecyclase domaindomain and and is is the the site site of of cGMP cGMP formation formation from from guanosine guanosine triphosphate triphosphate (GTP) (GTP) [18]. A[18]. single A single active active site issite formed is formed at the at theα/ βα/dimerβ dimer interface, interface, with with critical critical catalytic catalytic residuesresidues contributedcontributed by eacheach subunitsubunit [[19–22].19–22]. AA second,second, catalyticallycatalytically inactiveinactive nucleotidenucleotide bindingbinding site,site, termed the pseudosymmetric pseudosymmetric site, site, is is also also present present at at the the CAT CAT domain domain interface interface and and is isproposed proposed to be to involved be involved in allosteric in allosteric activity activity regulation regulation by adenosine by adenosine triphosphate triphosphate (ATP) (ATP)[23–25]. [23 –25].

Figure 1.1. An introduction toto sGC.sGC. ((AA)) sGCsGC domaindomain architecture.architecture. sGCsGC isis aa heterodimerheterodimer composedcomposed ofof homologous α and β subunits. Each subunit contains four domains: an N-terminal heme NO/O2- homologous α and β subunits. Each subunit contains four domains: an N-terminal heme NO/O2- binding (H-NOX) domain, a Per-Arnt-Sim (PAS) domain, a coiled coil (CC) domain, and a C-termi- binding (H-NOX) domain, a Per-Arnt-Sim (PAS) domain, a coiled coil (CC) domain, and a C-terminal nal catalytic (CAT) domain. The red trapezoid represents the heme cofactor found in the β H-NOX catalytic (CAT) domain. The red trapezoid represents the heme cofactor found in the β H-NOX domain. Residue numbering of the domains corresponds to the sequence of human sGC. (B) Typical domain.activity profile Residue of numbering sGC. sGC ofexhi thebits domains a low, corresponds basal level toof theactivi sequencety in the of humanabsence sGC. of NO (B) Typical(U: un- activityliganded). proﬁle Addition of sGC. ofsGC a single exhibits equivalent a low, basal of NO level (1-NO) of activity results in in the a modest absence increase of NO (U: in unliganded).activity. Full AdditionsGC activity of a is single obtained equivalent in the ofpr NOesence (1-NO) of a resultsmolar inexcess a modest of NO increase (xsNO). in Activity activity. Fullcan also sGC be activity stim- isulated obtained by the in small the presence molecule of 3-(5 a molar′-hydroxymethyl-2 excess of NO′ (xsNO).-furyl)-1-benzylindazole Activity can also (YC-1). be stimulated (C) UV-Visible by the smallabsorption molecule spectra 3-(5 of0-hydroxymethyl-2 ligand binding to0 -furyl)-1-benzylindazolethe sGC heme. Upon NO (YC-1).binding, ( Cthe) UV-Visible Soret peak absorptionshifts from spectra~430 to of399 ligand nm. The binding inset toshows the sGCthe Q-band heme. Uponregion NOof the binding, spectra. the Figure Soret adapted peak shifts from from reference ~430 to[11] 399 [Creative nm. The Commons inset shows Attribution the Q-band license] region. of the spectra. Figure adapted from reference [11] [Creative Commons Attribution license]. 3. Activity Profile of sGC 3. ActivityIn vitro, Proﬁle sGC exhibits of sGC a low basal level of activity in the absence of any ligand (Figure 1B) [23,26,27].In vitro, sGC An increase exhibits in a sGC low activity basal level is observed of activity upon in the the addition absence of of NO, any either ligand in (Figurethe form1B) of [NO23, 26gas,27 or]. through An increase the use in of sGC small activity molecule is observed NO donors, upon such the as addition NONOates of NO,[23,26–28]. either Interestingly, in the form ofthe NO extent gas of or enzyme through activation the use ofdepends small on molecule the relative NO donors,amount suchof NO as added. NONOates A modest [23,26 (~4-fold)–28]. Interestingly, increase in sG theC activity extent ofis observed enzyme activationin the presence depends of a onsingle the molar relative equivalent amount ofof NONO, added. which Ais modesttermed the (~4-fold) 1-NO increasestate (Figure in sGC 1B) activity [23,26,27]. is ob- In servedthis state, in theNO presenceforms a 5-coordinate of a single molarFe2+–NO equivalent complex ofwith NO, the which heme iscofactor termed of the the 1-NO β H- state (Figure1B) [23,26,27]. In this state, NO forms a 5-coordinate Fe 2+–NO complex with the heme cofactor of the β H-NOX domain, which is discernable by ultraviolet-visible (UV-Vis) absorption spectroscopy as a shift in the Soret band of the heme from ~430 nm 2+ (for the Fe unliganded species; the exact λmax depends on the species of sGC) to 399 nm (Figure1C) [ 29,30]. The activity of the 1-NO state accounts for, on average, ~15% of the Int. J. Mol. Sci. 2021, 22, 5439 3 of 18

maximal observed sGC activity, which is achieved in the presence of a stoichiometric excess of NO, and which is termed the excess-NO state (Figure1B) [ 23,26,27]. In this state, the heme remains as a 5-coordinate Fe2+–NO complex and the site (or sites) of additional NO binding remains unknown, although cysteine residues have been implicated as potential binding partners [27]. Notably, the additional NO present in the excess-NO state can be removed by dialysis or buffer exchange over a desalting column to yield the 1-NO state of the protein, indicating that the additional NO forms only a transient interaction with sGC. After removal of the excess NO, the enzyme returns to the 1-NO state activity level. In addition to the excess-NO state, a high level of sGC activity can also be achieved through the addition of small molecule stimulators to the 1-NO state of the protein. The prototypical sGC stimulator is the benzylindazol derivative YC-1 (3-(50-hydroxymethyl-20- furyl)-1-benzylindazole), which was ﬁrst identiﬁed in a screen of compounds for platelet inhibition [31]. Since its discovery, the structure of YC-1 has been used as a template for drug development efforts aimed at creating sGC stimulators, including for the drug riociguat (Bayer’s Adempas®) that is used in the treatment of pulmonary hypertension [9,32–35]. Addition of YC-1 to the 1-NO state of sGC leads to activity that is comparable to that of the excess-NO state (Figure1B) [27,36]. The existence of various activity states for sGC in vitro begs the question of physiological relevance—which activity states of sGC are relevant in vivo? In cells, the concentration of NO has been estimated to be in the range of 100–5000 pM [37], whereas estimates for the dissociation constant of NO from the sGC heme put the Kd in the low picomolar range, or lower [38]. Based on these considerations, the unliganded state of sGC is unlikely to exist in cells. Therefore, the physiologically relevant forms of sGC likely cycle between a 1-NO state and an excess-NO activated state. This inference is supported by two independent studies done in vein and aortic endothelial cells in which YC-1 was shown to stimulate cellular cGMP production to a high level, but not in the presence of inhibitors of nitric oxide synthase (NOS) [27,39]. The underlying assumption is that when NOS is inhibited, the intracellular NO concentration is low enough that sGC exists in the unliganded state. These results are consistent with the in vitro observations that YC-1 stimulates the 1-NO state of the protein to a high activity level, whereas the unliganded state is stimulated to a much lesser extent (Figure1B).

4. Crystal Structures of Individual sGC Domains Although more than four decades of work have revealed much about the biochemistry of sGC, obtaining a structure of the full-length enzyme proved elusive. In lieu of such information, structural insights on sGC came from crystal structures of individual sGC domains and their homologs, as summarized below.

4.1. The H-NOX Domain Efforts to better understand the effect of NO binding to sGC turned to bacterial homologs of the β H-NOX domain. In aerobic and facultative anaerobic bacteria, NO-sensing H-NOXs are found as standalone proteins that interact with cognate signaling partners, usually a histidine kinase or a diguanylate cyclase [40,41]. In contrast, obligate anaerobes contain O2-sensitive H-NOX domains fused to C-terminal effector domains, generally a methyl-accepting chemotaxis protein [40,42]. Both NO-binding and O2-binding H-NOX domains have been studied in detail structurally and spectroscopically to provide insight into the function of this domain in sGC. The ﬁrst H-NOX structure to be solved was of the O2-binding H-NOX domain from the thermophilic anaerobe Caldanaerobacter subterraneous (Cs) (previously known as Thermoanaerobacter tengcongensis)[43,44]. The structure revealed the H-NOX domain to consist of seven α-helices (labeled αA–G) and a four-stranded antiparallel β-sheet (with strands labeled β1–4). The b-type heme cofactor is found sandwiched between the N- and C-terminal subdomains of the protein and is ligated by a conserved histidine residue, termed the proximal histidine, which is located on the αF helix of the protein. The N- and C-terminal H-NOX subdomains are termed distal and Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 18

(Cs) (previously known as Thermoanaerobacter tengcongensis) [43,44]. The structure revealed the H-NOX domain to consist of seven α-helices (labeled αA–G) and a four- stranded antiparallel β-sheet (with strands labeled β1–4). The b-type heme cofactor is Int. J. Mol. Sci. 2021, 22, 5439 4 of 18 found sandwiched between the N- and C-terminal subdomains of the protein and is ligated by a conserved histidine residue, termed the proximal histidine, which is located on the αF helix of the protein. The N- and C-terminal H-NOX subdomains are termed distal proximal,and proximal, respectively, respectively, with with the proximal the proximal subdomain subdomain taking taking its name its name from from the presencethe pres- ofence the of proximal the proximal histidine histidine residue residue within with thein subdomain. the subdomain. The distalThe distal subdomain subdomain consists con- ofsistsα-helices of α-helices A–E andA–E theand proximal the proximal subdomain subdomain is composed is composed of helices of helicesαF and αF αandG andαG and the four-strandedthe four-strandedβ-sheet. β-sheet. SubsequentSubsequent crystalcrystal structuresstructures ofof thethe NO-bindingNO-binding H-NOXH-NOX fromfromShewanella Shewanella oneidensisoneidensis ((SoSo)) inin the the unliganded unliganded and and NO-bound NO-bound states states revealed revealed the the structural structural effects effects of gasof gas binding binding to theto the protein protein [45]. [45]. Upon Upon NO binding,NO binding, the protein the protein undergoes undergoes a global a conformationalglobal conformational change ◦ inchange which in the which distal the subdomain distal subdomain rotates ~4 rotatesrelative ~4° relative to the proximal to the proximal subdomain subdomain (Figure 2(Fig-A ). Thisure 2A). rotation This is rotation centered is on centered two conserved on two glycineconserved residues glycine within residues the αwithinD and theαG α helices,D and calledαG helices, the glycine called hinge the glycine [45–47 hinge]. A similar [45–47]. ligation A similar state—induced ligation state—induced conformational conforma- change wastional later change observed was inlater the observed O2-binding in CstheH-NOX, O2-binding and itCs has H-NOX, been suggested and it has that been this suggested rotation ofthat the this distal rotation subdomain of the distal may besubdomain a general may feature be a of general H-NOXs feature which of couldH-NOXs play which a key could role inplay signaling a key role [46 ].in signaling [46].

FigureFigure 2.2. NO binding induces induces conformational conformational changes changes in in SoSo H-NOX.H-NOX. (A (A) Overall) Overall structures structures of ofSo SoH- H-NOXNOX in inthe the unliganded unliganded (light (light green, green, PDB PDB ID 4U99 ID 4U99 [45]) [ 45and]) andNO-bou NO-boundnd (dark (dark green, green, PDB PDBID 4U9B ID [45]) states. NO binding induces rotation of the αF helix and a shift of the distal subdomain away 4U9B [45]) states. NO binding induces rotation of the αF helix and a shift of the distal subdomain from the heme binding pocket, as indicated by the black arrows. Structures were aligned by least away from the heme binding pocket, as indicated by the black arrows. Structures were aligned by squares fitting by the β-sheet region of the proximal subdomain (r.m.s.d. = 0.55 Å for 62 Cα atoms). β α least(B) Zoomed squares in ﬁtting view byof the the heme-sheet cofactor region showing of the proximal cleavage subdomainof the iron–His103 (r.m.s.d. bond = 0.55 as viewed Å for 62 down C atoms).the axis (ofB )the Zoomed αF helix. in viewProtein of is the shown heme in cofactor ribbon showingrepresentation cleavage with of the the heme iron–His103 in sticks bond and the as viewediron center down in spheres. the axis ofHis103 the α Fis helix.shown Protein in sticks is.shown Sticks and in ribbon spheres representation are colored with with C the in hemegreen,in N sticksin blue, and O thein red, iron and center Fe in orange. spheres. The His103 N- and is shown C-termini in sticks. are labeled Sticks andand spheres shown areas small colored spheres with Cin inpanel green, A. N in blue, O in red, and Fe in orange. The N- and C-termini are labeled and shown as small spheres in panel A. In addition to the overall conformational change, the structures also show NO-in- ducedIn rupture addition of to the the iron–histidine overall conformational bond between change, the the heme structures cofactor also and show the NO-inducedprotein, con- rupturesistent with of the previous iron–histidine spectroscopic bond between data [45] the. hemeThis bond cofactor cleavage and the is protein,accompanied consistent by a withslight previous distortion spectroscopic of the αF helix data such [45]. that This the bond Cα atom cleavage of the is accompaniedproximal histidine by a slightis dis- distortionplaced by of~2 theÅ andαF the helix side such chain that adopts the C anα atom alternative of the rotamer proximal conformation. histidine is displaced Together, bythese ~2 movements Å and the side result chain in an adopts apparent an alternative ~90° rotation rotamer of the conformation.histidine side chain Together, away these from ◦ movementsits position when result it in is anligated apparent to the ~90hemerotation (Figure 2B). of the Notably, histidine in the side structure chain away of the from NO- itsbound position So H-NOX, when itNO is ligatedwas observed to the hemebound (Figure to the proximal2B). Notably, side of in the the heme, structure rather of thethan NO-boundto the distalSo sideH-NOX, as had NObeen was expected. observed Subsequent bound to spectroscopic the proximal work, side of however, the heme, revealed rather thanthis phenomenon to the distal sideto be as a hadresult been of the expected. large excess Subsequent of NO used spectroscopic in the crystallization work, however, con- revealedditions and this that phenomenon under standard to be a solution result of conditions the large excess NO primarily of NO used binds in the to crystallizationthe distal side conditionsof the heme and [48]. that under standard solution conditions NO primarily binds to the distal side of the heme [48].

4.2. The PAS and CC Domains PAS and CC domains are ubiquitous and versatile structural motifs that are found across domains of life and that often function together in a signaling capacity. PAS domains

are named for the proteins Per, Arnt, and Sim, the three proteins in which homologous sequence regions, now recognized as the PAS domain, were ﬁrst identiﬁed [49]. There are now over 40,000 annotated PAS domains in the Pfam database [50] and, despite having Int. J. Mol. Sci. 2021, 22, 5439 5 of 18

low sequence similarities on average, these domains share a common core fold consisting of a five-stranded antiparallel β-sheet flanked by several α-helices [15]. A subset of PAS domains bind cofactors and/or exogenous ligands and serves in a signaling capacity as direct sensors. For the majority of PAS domains, however, no ligand has been identified and these domains are thought to play a role in signal transduction and modulating protein–protein interactions [15]. Indeed, the sGC PAS domains are not known to bind a ligand but do contribute to dimerization. The first structural information on the sGC PAS domains came from a crystal structure of a histidine kinase PAS domain that is highly homologous to the sGC domains [51]. This structure showed that the PAS domains in sGC likely form a parallel dimer through a conserved dimerization motif that is common to many PAS domains [51,52]. Subsequently, a crystal structure of the α PAS domain from the Manduca sexta (moth) sGC was determined [53]. How the PAS domains may contribute to signal transduction in sGC is unclear from the structures, although rearrangement of internal hydrogen bonding networks has been implicated in structurally similar PAS dimers [52]. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) revealed NO- induced differences in H/D exchange rates in parts of the PAS domains in the full-length Rattus norvegicus (rat) sGC, consistent with the idea that perturbations in this region occur upon activation [54]. As the name implies, CC domains are composed of two or more α-helices that intertwine, forming a coil of coils. These domains are found in a wide range of proteins and serve in a variety of functions. As in sGC, CC domains are often found C-terminal to PAS domains, where they are thought to serve as an intermediary signal transduction module between the PAS domain and a connected effector domain, this effector domain being the CAT domain in sGC [15]. Previous structural information on the sGC CC domain came from a crystal structure of the isolated rat β CC domain [55]. Efforts to obtain a structure of the α/β CC heterodimer in this study were stymied by an inability to express the α CC domain. The structure of the β CC showed a tetrameric assembly composed of two CC homodimers, and within each dimer, the two helices interact in an unexpected anti-parallel fashion. Based on sequence considerations and modeling of the full-length sGC structure, the authors reasoned that the anti-parallel arrangement likely does not reflect the true structure of the α/β CC heterodimer, which they concluded is more likely to be oriented in a parallel manner. This conclusion was supported by a later study in which chemical crosslinking between lysine residues in the moth sGC was more consistent with a parallel arrangement of the CC domains [56]. Similar to the PAS domains, the CC domains of the full-length rat sGC also exhibit changes in H/D exchange rates upon NO binding, consistent with alterations in this portion of the structure upon activation [54].

4.3. The CAT Domain The ﬁrst nucleotide cyclase CAT domains to be structurally characterized were those of adenylate cyclases (ACs) [57–59]. The AC CAT domain is comprised of a large eight- stranded mixed β-sheet surrounded by ﬁve α-helices (Figure3A). Two CAT domain monomers intertwine in a head-to-tail fashion to form a wreath-like dimer (Figure3B). The active site is found in a cleft at the dimer interface and is composed of residues that are contributed by each subunit. This architecture gives rise to two nucleotide/ligand binding sites at the dimer interface, which translates to the presence of two active sites in AC homodimers and of one active site and one pseudosymmetric site in AC heterodimers. In heterodimers, the pseudosymmetric site lacks the residues necessary for catalysis and is proposed to be involved in activity regulation [57,58]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 18

Int. J. Mol. Sci. 2021, 22, 5439 6 of 18 heterodimers, the pseudosymmetric site lacks the residues necessary for catalysis and is proposed to be involved in activity regulation [57,58].

Figure 3.3. CrystalCrystal structures structures of of AC AC CAT CAT domains domains serve serve as a as prototype a prototype for GC for CAT GC domains.CAT domains. (A) Struc- (A) tureStructure of the of AC the CAT AC domain CAT domain monomer monomer (PDB ID (PDB 1CJK ID [59 1CJK]). Secondary [59]). Secondary structure structure elements elements are labeled are accordinglabeled according to conventions to conventions from the from literature the literatur [57,58].e Protein[57,58]. isProtein shown is inshown ribbon in representation ribbon representa- and coloredtion and in colored rainbow in moderainbow from mode N- tofrom C-terminus N- to C-terminus (termini are(termini shown are as shown small spheresas small andspheres labeled) and labeled) with the N-terminus in blue and the C-terminus in red. (B) Structure of the AC CAT dimer with the N-terminus in blue and the C-terminus in red. (B) Structure of the AC CAT dimer with with ATP and two Mg2+ ions bound. Each monomer is shown as in panel A with monomer A at 50% ATP and two Mg2+ ions bound. Each monomer is shown as in panel A with monomer A at 50% transparency. Secondary structural elements that shift upon activation are labeled. transparency. Secondary structural elements that shift upon activation are labeled. For many years, AC CAT domain structures served as a basis for understanding the For many years, AC CAT domain structures served as a basis for understanding structure of GCs, given their high sequence similarities. Indeed, homology models of the the structure of GCs, given their high sequence similarities. Indeed, homology models bovine and rat sGC CAT domains that were made based on AC CAT domains were used of the bovine and rat sGC CAT domains that were made based on AC CAT domains wereto identify used tokey identify residues key involved residues in involved catalysis inand catalysis substrate and specificity substrate [60,61]. specificity Using [60 these,61]. Usingmodels these as a modelsguide, the as aauthors guide, in the one authors study inwere one able study to werealter the able substrate to alter thespecificity substrate of specificitythe rat sGC, of therendering rat sGC, it rendering capable of it capableperformi ofng performing AC activity AC [61]. activity More [61 recently,]. More recently, crystal crystalstructures structures have been have solved been solvedof both of homo- both homo- and hetero-dimeric and hetero-dimeric GC CAT GC CATdomains, domains, con- confirmingfirming that that their their structures structures are arein fact in fact similar similar to those to those of ACs of ACs [19–22]. [19–22 ]. To date,date, allall GCGC CATCAT domaindomain crystalcrystal structuresstructures representrepresent inactiveinactive conformationsconformations inin whichwhich thethe activeactive sitessites areare not appropriately orientedoriented forfor substratesubstrate bindingbinding andand subsequentsubsequent catalysiscatalysis [[19–22].19–22]. Mechanisms for activation have been proposedproposed basedbased onon structuresstructures ofof ACAC CATCAT domainsdomains thatthat appearappear toto representrepresent catalyticallycatalytically competentcompetent conformationsconformations [[59].59]. InIn general, these mechanisms entail a rotation of oneone subunitsubunit towardstowards thethe otherother inin aa clampclamp likelike motionmotion thatthat involvesinvolves movementmovement ofof thethe ββ7–7–ββ8 loop andand heliceshelices αα11 andand αα22 [[20–22].20–22]. InIn additionaddition toto thisthis large-scalelarge-scale conformationalconformational change,change, smallersmaller changeschanges withinwithin thethe activeactive sitesite openopen up the the substrate substrate binding binding site. site. In Inparticular, particular, helix helix α4 αshifts4 shifts away away from from the central the central cleft cleftto make to make room room for substrate. for substrate. Full closure Full closure of the of active the active site is sitethought is thought to occur to occurafter initial after initialsubstrate substrate binding binding in a step in athat step brings that brings key amino key amino acid residues acid residues and the and nucleotide the nucleotide sub- substratestrate into into favorable favorable alignment alignment for for catalysis catalysis [19,20]. [19,20]. It It has has been been suggested suggested that substratesubstrate discriminationdiscrimination betweenbetween GTP GTP and and ATP ATP occurs occurs during during this this closure closure step, step, as residues as residues involved in- involved base in recognition base recognition are brought are brought into position into position to optimally to optimally hydrogen hydrogen bond with bond either with guanineeither guanine or adenine or adenine [19]. [19].

5.5. Low-Resolution Structures of Full-Length sGCsGC The ﬁrstfirst directdirect insightinsight intointo thethe structurestructure ofof full-lengthfull-length sGCsGC camecame inin 20142014 withwith thethe reportreport of low-resolution low-resolution negative negative stain stain electr electronon microscopy microscopy (EM) (EM) envelopes envelopes of the of therat raten- enzymezyme [62]. [62 Three-dimensional]. Three-dimensional EM EM maps maps of th ofe the uranyl uranyl formate-stain formate-staineded particles particles were were ob- obtainedtained through through random random conical conical tilt tilt (RCT) (RCT) reconstructions reconstructions and and ranged ranged in resolution fromfrom 2525 toto 4040 Å.Å. TheThe mapsmaps revealedrevealed aa dumbbell-shapeddumbbell-shaped structurestructure composedcomposed ofof twotwo globularglobular lobeslobes connectedconnected byby aa thin,thin, stalk-likestalk-like regionregion ofof density.density. TheThe largerlarger ofof thethe twotwo globularglobular lobeslobes waswas assignedassigned asas thethe H-NOX/PASH-NOX/PAS bundle,bundle, withwith the the two two PAS PAS domains domains interacting interacting to to form form a dimer interface and the two H-NOX domains ﬂanking either side of this central PAS dimer. The stalk-like structure extended from the PAS dimer and was assigned as the CC domains,

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running in a parallel orientation into the smaller globular lobe, which was assigned as the CAT domain dimer. The single-particle classification and alignment in this study yielded a total of 94 3D reconstructions of the unliganded rat sGC that suggested significant conformational flexibility [62]. The reconstructions ranged from fully extended states, in which the H- NOX/PAS bundle and the CAT domains were separated by 40 Å, to more compact states, in which connective density was observed between the β H-NOX and CAT domains. The two ends of the CC dimer were described as pivot points around which these conformational changes take place. Notably, this conformational flexibility was independent of the enzyme ligation state, as structural analysis performed on NO and GTP analog-bound sGC revealed similar distributions of conformational states [62]. This low-resolution analysis of full-length sGC unveiled for the first time the overall shape and general domain organization of the enzyme, however, the mechanism of allosteric activity regulation remained unclear. It was not until the determination of higher resolution structures that these details began to become more apparent.

6. High-Resolution Cryo-EM Structures of Full-Length sGC Within the last decade, the so-called “Resolution Revolution” [63] in single-particle cryo-EM has ushered in a new era in structural biology, and in 2019, two independent reports of high-resolution full-length sGC structures were published [64,65]. In the ﬁrst report, Horst et al. presented structures of the sGC from Manduca sexta (moth) in the unactivated, Fe2+-unliganded state and in the excess-NO and YC-1-bound activated state to 5.1 and 5.8 Å resolution, respectively [64]. The second report, by Kang et al., presented several structures of the Homo sapiens (human) sGC in different states: the Fe2+-unliganded state (to 4.0 Å resolution), the Fe3+-unliganded state (3.9 Å resolution), the excess-NO activated state with bound Mg2+ and GMPCPP (3.8 Å resolution), and the β H105C variant of the enzyme (6.8 Å resolution) [65]. These two sGC isoforms share 38% sequence identity in the α subunits, 61% identity in the β subunits, and both exhibit the three-state activity proﬁle discussed above [64,66,67]. Consistent with the similarities in sequence and activity, the structures of the moth and human enzymes are very alike and both sGCs were found to undergo a dramatic conformational change upon ligand binding and activation, as will be discussed in detail below. The Fe3+-unliganded form of human sGC was found to be structurally similar to the Fe2+-unliganded form and the β H105C variant, in which the heme-ligating histidine is replaced with cysteine, was found to lack the heme cofactor and to be similar in both structure and activity to the activated state of the enzyme [65].

6.1. General Architecture of Full-Length sGC in the Unactivated and Activated States Consistent with the low-resolution, negative stain EM reconstructions, the high- resolution cryo-EM data reveal an oblong particle composed of two distinct lobes connected through a stalk-like density. The N-terminal regulatory lobe consists of the α and β H- NOX domains and the α/β PAS dimer (Figure4). The two CC domains extend from the PAS dimer to the C-terminal regulatory lobe, which contains the α/β CAT domain dimer (Figure4). Int. J. Mol. Sci. 2021, 22, 5439 8 of 18 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 18

FigureFigure 4. 4.Activation Activation of of sGC sGC induces induces a a large-scale large-scale conformational conformational change change involving involving straightening straightening of of thethe CC CC domains. domains. (A (A) Views) Views of of the the unactivated unactivated conformation conformation of sGCof sGC (PDB (PDB ID 6JT0ID 6JT0 [65 ]).[65]). (B) Views(B) Views of theof activatedthe activated conformation conformation of sGC of sGC (PDB (PDB ID 6JT2 ID [6JT265]). [65]). Protein Protein is shown is shown in ribbon in ribbon representation representation with with domains colored as indicated on the figure. The regions of the CC domains that bend and domains colored as indicated on the ﬁgure. The regions of the CC domains that bend and straighten straighten are colored purple. The heme cofactor in the β H-NOX domain is shown in sticks with C are colored purple. The heme cofactor in the β H-NOX domain is shown in sticks with C in pink, N in pink, N in blue, O in red, and Fe in orange. in blue, O in red, and Fe in orange. In the basal, unactivated state, sGC adopts a contracted conformation in which the β In the basal, unactivated state, sGC adopts a contracted conformation in which the H-NOX domain is in close proximity to, although not directly interacting with, the β CAT β H-NOX domain is in close proximity to, although not directly interacting with, the domain (Figure 4A). This bent conformation appears to be achieved through an unex- β CAT domain (Figure4A). This bent conformation appears to be achieved through an pected bending of the CC domains (highlighted in purple in Figure 4A). Following the unexpected bending of the CC domains (highlighted in purple in Figure4A). Following thePAS PAS domain domain dimer, dimer, the the α andα and β CCβ CC domains domains form form helical helical segments segments of of3 and 3 and 2 turns, 2 turns, re- respectively,spectively, followed followed by loop regions thatthat leadlead intointo twotwo longerlonger helical helical segments segments at at an an angle angle ofof ~90 ~90°◦ fromfrom thethe initial short helices. helices. Upon Upon ac activation,tivation, the the conformation conformation of ofthe the protein protein ex- extendstends as as the the regulatory regulatory lobe lobe swings swings through through a ro atation rotation of of~70° ~70 relative◦ relative to the to thecatalytic catalytic lobe lobe(Figure (Figure 4B).4 B).In Inthe the activated activated state, state, the the portions portions of of the the CC domainsdomains thatthat areare bent bent in in the the unactivatedunactivated state state havehave adopted helical folds folds to to form form two two long, long, continuous continuous helices helices from from the thePAS PAS dimer dimer through through to to the the CAT CAT dimer, dimer, resulting in thethe overalloverall extendedextended conformation conformation (Figure(Figure4B). 4B). Interestingly,Interestingly, the the structures structures do do not not indicate indicate that that there there is is any any direct direct contact contact between between thethe regulatory regulatory and and catalytic catalytic lobes. lobes. Such Such direct direct contact contact had had previously previously been been suggested suggested as as thethe means means of of activity activity regulation regulation in in sGC, sGC, with with the theβ βH-NOX H-NOX domain domain interacting interacting with with the the CAT domains in an inhibitory fashion that was relieved upon NO binding [18,68]. Instead,

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it seems that the activation mechanism involves more subtle allosteric changes that are CAT domains in an inhibitory fashion that was relieved upon NO binding [18,68]. Instead, communicated through the length of the protein. The bending and straightening of the it seems that the activation mechanism involves more subtle allosteric changes that are CC domains appears to be a key conformational change in this process, although the spe- communicated through the length of the protein. The bending and straightening of the CC cific amino acid interactions that lead to this change remain unclear. domains appears to be a key conformational change in this process, although the specific The high-resolution structures of full-length sGC in both the unactivated and acti- amino acid interactions that lead to this change remain unclear. vated states provide a first exciting look at the structural details of this important enzyme. The high-resolution structures of full-length sGC in both the unactivated and activated They also set the stage for future experiments aimed at further understanding the mech- states provide a first exciting look at the structural details of this important enzyme. They anism of allosteric, intramolecular signal transduction. Aspects of individual domains that also set the stage for future experiments aimed at further understanding the mechanism have been revealed by these new structures will be discussed in more detail below. The of allosteric, intramolecular signal transduction. Aspects of individual domains that have higher resolution of the human sGC structures allowed visualization of many amino acid been revealed by these new structures will be discussed in more detail below. The higher side chains throughout the electron density maps, and so references to residue numbers resolution of the human sGC structures allowed visualization of many amino acid side willchains reflect throughout the numbering the electron of the densityhuman maps,sGC sequence. and so references to residue numbers will reflect the numbering of the human sGC sequence. 6.2. The α H-NOX Domain 6.2. The α αH-NOX H-NOXDomain domain of sGC has long been an enigma. It has previously been shown to be Theunnecessaryα H-NOX for domain dimerization of sGC hasor activity long been regulation, an enigma. and It yet has is previously present in been all known shown sGCsto be [14]. unnecessary Despite sharing for dimerization sequence orsimilarity activity with regulation, the β H-NOX and yet domain is present (Figure in all 5A), known the αsGCs H-NOX [14]. does Despite not sharingbind heme, sequence and has similarity previo withusly been the β termedH-NOX a domain “pseudo–H-NOX” (Figure5A), thefor thisα H-NOX reason does[14,54]. not Prior bind to heme, the high-resolution and has previously full-length been termed sGC structures, a “pseudo–H-NOX” no structural for informationthis reason [on14 ,the54]. α Prior H-NOX to the domain high-resolution was available, full-length due at least sGC structures,in part to an no inability structural to expressinformation the isolated on the domainα H-NOX in a domain soluble wasform available, [18]. Explanations due at least for why in part this to domain an inability does notto expressbind heme the isolatedwere based domain on sequence in a soluble consi formderations—the [18]. Explanations heme ligating for why histidine this domain resi- duedoes is not missing bind hemein the werehuman based sGC on α sequencesequence, considerations—thewhereas a conserved heme YxSxR ligating motif histidinethat sta- bilizesresidue the is missingheme propionate in the human chains sGC in αheme-bsequence,inding whereas H-NOXs a conserved is missing YxSxR in the motifsequence that ofstabilizes the moth the sGC heme α subunit propionate (Figure chains 5A). in heme-bindingIt was reasonably H-NOXs assumed is missing that the in lack the sequence of these residuesof the moth must sGC be theα subunit reason (Figurethat the5 A).α H-NOX It was reasonablydoes not bind assumed heme. thatWith the structures lack of these now inresidues hand, however, must be the the reason inability that to the bindα H-NOX heme becomes does not clear: bind an heme. N-terminal With structures extension now (res- in idueshand, α however,-69–85, α the-1–68 inability are disordered) to bind heme of the becomes α H-NOX clear: runs an N-terminalinto the heme extension pocket, (residues occlud- ingα-69–85, heme bindingα-1–68 are(Figure disordered) 5B). Although of the theα H-NOX function runs of this into domain the heme remains pocket, elusive, occluding there isheme now bindinga more complete (Figure5B). understanding Although the of function why sGC of contains this domain only remains one heme elusive, per heterodi- there is mer.now a more complete understanding of why sGC contains only one heme per heterodimer.

FigureFigure 5. 5. AnAn N-terminal N-terminal extension extension of of the the αα H-NOXH-NOX domain domain occludes occludes heme heme binding. binding. ( (AA)) Sequence Sequence alignment alignment of of the the αα andand ββ H-NOXH-NOX domains domains from from human human ( (HsHs)) and and moth moth ( (MsMs)) sGCs. sGCs. The The αα andand ββ H-NOXH-NOX domains domains share share 20 20 and and 18% 18% sequence identityidentity for for the the human human and and moth moth sequences, sequences, re respectively.spectively. The The N-termin N-terminalal extension of the α H-NOXH-NOX domain domain that that is is visible visible in the human sGC EM structure is highlighted in pink. The heme-ligating histidine (β-His105) and the YxSxR motif of the in the human sGC EM structure is highlighted in pink. The heme-ligating histidine (β-His105) and the YxSxR motif of β H-NOX domain are highlighted in blue and green, respectively. Functionally conserved residues are in bold font. (B) the β H-NOX domain are highlighted in blue and green, respectively. Functionally conserved residues are in bold font. Superposition of the α and β H-NOX domains from the unactivated human sGC structure (PDB ID 6JT0 [65]). The N- B α β terminal( ) Superposition helical extension of the ofand the α H-NOXH-NOX domainsdomain is from shown the in unactivated pink and clashes human with sGC the structure heme in (PDB the β ID H-NOX 6JT0 [ 65domain.]). The StructuresN-terminal were helical aligned extension by secondary of the α H-NOX structure domain matching is shown (r.m.s.d. in pink = 1.88 and Åclashes for 121 withresidues the heme aligned). in the Proteinβ H-NOX is shown domain. as inStructures Figure 4 werewith the aligned heme by cofactor secondary C atoms structure in light matching blue. Th (r.m.s.d.e N- and = 1.88C-terminal Å for 121 residues residues of each aligned). domain Protein are labeled is shown and as shownin Figure as 4small with spheres. the heme cofactor C atoms in light blue. The N- and C-terminal residues of each domain are labeled and shown as small spheres.

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6.3. The β H-NOX Domain In contrast to the α H-NOX domain, the β H-NOX domain and its bacterial homologs have been well-characterized in previous studies. Much of the interest in the β H-NOX domain lies in the fact that it contains the binding site for the first equivalent of NO and is therefore an essential component of NO-dependent activity regulation. Previous structural work on bacterial H-NOX homologs revealed a ligation-state dependent conformational change in which the distal subdomain rotates relative to the proximal subdomain upon NO binding, as discussed above (Figure2A) [ 45]. In the cryo-EM structures of full-length sGC, relative shifts between the two subdomains are less apparent (Figure6A). When the β H-NOX domains of the unactivated and activated human sGC structures are aligned by the β sheet region of the proximal subdomain (equivalent to the alignment performed for the So structures in Figure2A), helices αA–C of the distal subdomain are nearly superimposable (Figure6A). Instead, a lateral shift is observed in the αF helix (residues β-94–112), which contains the heme ligating histidine residue (β-His105) (Figure6A). This shift is likely to be induced by rupture of the iron–histidine bond, which occurs upon NO binding, and stands in contrast to the rotation of the αF helix that was observed in the structures of So H-NOX. Also in contrast to the So H-NOX structures is the relative positioning of β-His105 (His103 in So H-NOX) in the unactivated (unliganded) and activated (NO-bound) structures. Rather than the histidine side chain flipping away from the heme, the rotamer conformation of β-His105 is maintained following iron–histidine bond cleavage, while the Cα atom of the residue shifts by 2.1 Å (Figure6A). Notably, if β-His105 were to flip upon NO binding as it does in So H-NOX, it would clash with β-Arg258 in the β PAS domain of the activated conformation of full-length sGC. Together, these differences in NO-induced structural changes suggest that the mechanism of communicating the heme ligation state is different for isolated H-NOX domains than it is for full-length sGC. Structures of bacterial H-NOX domains in complex with their cognate signaling partners would provide more insight into these differences. One interesting aspect of the NO-induced shift of the αF helix is that it results in a loss of hydrogen bonding interactions between residues on the C-terminal end of the αF helix and residues within the α CC domain (Figure6B). In particular, β-Thr110 appears to make contacts with α-Gln418 and α-Arg428 in the structure of the unactivated human sGC (Figure6B). Additionally, β-His107 contacts α-Glu417 (Figure6B). Notably, α-Gln418 and α-Arg428 bookend the bend in the α CC domain. Thus, one mechanism for NO-induced sGC activation may be that the shift of the αF helix disrupts contacts with these residues, allowing the α CC to straighten. The residues β-His107, β-Thr110, α-Glu417, α-Gln418, and α-Arg428 are all highly conserved in over 200 sGC sequences, underscoring their potential functional relevance. In previous biochemical work, a β-T110A variant of rat sGC was shown to exhibit higher basal activity than WT, consistent with a role for β-Thr110 in stabilizing the unactivated conformation of the protein [69]. A separate study, however, showed that the β-T110R variant had comparable activity to WT in enriched lysate assays, and so the exact function of this residue remains poorly understood [70]. Higher resolution structures and/or further mutagenesis studies would provide more insight into the role of β-Thr110 and the residues that it appears to interact with in the unactivated conformation. Also on the αF helix is β-Asp106, which is also highly conserved in over 200 sGC sequences and has been implicated in sGC activation. Mutation of this residue to alanine or lysine has been shown to result in an sGC variant that cannot be fully activated by excess NO [65,70]. Interestingly, β-Asp106 appears to participate in a network of charge– charge interactions in both the unactivated and activated states, although the interaction partners are different in each case (Figure6C,D). In the unactivated conformation, β-Asp106 contacts β-Arg258 in the β PAS domain, which in turn also contacts β-Asp374 in the β CC domain (Figure6C). Activation of sGC and shifting of the αF helix disrupts these contacts, and in the activated state β-Asp106 contacts both β-Arg305 and β-Lys307 in the β PAS domain; β-Lys307 additionally interacts with α-Asp411 in the α CC (Figure6D). Following disruption of its interaction with β-Asp106, β-Arg258 forms an interaction with Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 18

FigureFigure 6.6. Structural rearrangements rearrangements in in the the ββ H-NOXH-NOX αFα Fhelix helix occur occur upon upon activation. activation. (A) (Superposi-A) Super- positiontion of the of theβ H-NOXβ H-NOX domains domains in the in unactivated the unactivated (grey, (grey, PDB PDB ID 6JT0 ID 6JT0[65]) [65and]) andactivated activated (light (light blue, PDB ID 6JT2 [65]) states. Activation induces a lateral shift of the αF helix, as indicated by the black blue, PDB ID 6JT2 [65]) states. Activation induces a lateral shift of the αF helix, as indicated by the black arrow. Structures were aligned by least squares ﬁtting by the β-sheet region of the proximal subdomain (r.m.s.d. = 0.70 Å for 67 Cα atoms). (B) In the unactivated state, residues within the αF

helix make contact with residues in the α CC domain. (C) In the unactivated state, β-Asp106 on the αF helix participates in an interaction network with β-Arg258 in the β PAS domain and β-Asp374 in the β CC domain. The α sGC subunit has been omitted from this panel for clarity. (D) In the activated state, residues along the αF helix make different contacts than in the unactivated state. Domains are colored as in Figure4 with the heme C atoms in light blue. Amino acid side chains are shown as sticks with C in respective domain colors, N in blue, and O in red. Hydrogen bonding interactions are shown as black dashes. The N- and C-terminal residues of the β H-NOX domain are labeled and shown as small spheres in panel A.

6.4. The PAS Domains The α and β PAS domains of sGC form a parallel dimer, as predicted by the crystal structure of the homologous histidine kinase PAS domain [51]. The dimer interface is stabilized by a conserved dimerization motif consisting of hydrophobic interactions along an N-terminal amphipathic helix (residues α-279–286, β-210–217) as well as an adjacent β-strand (residues α-378–382, β-318–322) (Figure7)[ 51,52]. The overall structure of the sGC PAS dimer changes very little upon activation (Figure7). It was previously suggested that signal transduction in similar PAS domain dimers occurred through alterations of internal hydrogen bonding networks [52]. What these alterations are in sGC is unclear from the structures and how the PAS domains might play a role in signaling remains ambiguous. It may be that the PAS domains play a dynamic role in facilitating structural transitions Int.Int. J. J. Mol. Mol. Sci. Sci.2021 2021,,22 22,, 5439 x FOR PEER REVIEW 12 ofof 1818

thatthat areare notnot apparentapparent inin thethe staticstatic endpointendpoint snapshotssnapshots capturedcaptured byby EM.EM. Alternatively,Alternatively, the the rolerole ofof thethe PASPAS domains domains could could be be mainly mainly structural structural in in facilitating facilitating dimerization. dimerization.

FigureFigure 7.7.The The twotwo sGCsGC PASPAS domains domains interact interact via via a a conserved conserved dimerization dimerizationmotif motifand and change changevery very littlelittle uponupon activation. Superpositio Superpositionn of of the the unactivated unactivated (grey, (grey, PDB PDB ID ID6JT0 6JT0 [65]) [65 and]) andactivated activated (colored as in Figure 4, PDB ID 6JT2 [65]) sGC PAS domains. The hydrophobic residues (α-I277, α-F282, (colored as in Figure4, PDB ID 6JT2 [ 65]) sGC PAS domains. The hydrophobic residues (α-I277, α-F286, α-L380, α-L382; β-I208, β-F213, β-F217, β-L320, β-L322) that interact at the dimer interface α-F282, α-F286, α-L380, α-L382; β-I208, β-F213, β-F217, β-L320, β-L322) that interact at the dimer are shown as sticks. Structures were aligned by secondary structure matching over both chains interface(r.m.s.d. are= 0.96 shown Å for as 234 sticks. residues Structures aligned). were The aligned N- and by secondaryC-terminal structure residuesmatching of each domain over both are chainslabeled (r.m.s.d. and shown = 0.96 as Å smallfor 234 spheres residues (C-terminal aligned). re Thesidue N- labels and C-terminal have been residuesomitted from of each the domain bottom arepanel). labeled and shown as small spheres (C-terminal residue labels have been omitted from the bottom panel). 6.5. The CC Domains 6.5. The CC Domains The most obvious conformational change that takes place upon sGC activation occurs The most obvious conformational change that takes place upon sGC activation occurs in the CC domains. In the unactivated structure, each CC domain is divided into two in the CC domains. In the unactivated structure, each CC domain is divided into two nearly perpendicular helical segments connected by a loop region that straightens upon nearly perpendicular helical segments connected by a loop region that straightens upon activation to form two long helices (Figure 8A). These bent loop regions comprise residues activation to form two long helices (Figure8A). These bent loop regions comprise residues α-419–427 and β-355–359. The higher resolution (4.0 Å) structure of the unactivated hu- α-419–427 and β-355–359. The higher resolution (4.0 Å) structure of the unactivated human man sGC allowed for more accurate modeling of the CC register in this region due to the sGC allowed for more accurate modeling of the CC register in this region due to the ability to observe electron density for many of the side chains [65]. Consistent with the ability to observe electron density for many of the side chains [65]. Consistent with the adoption of secondary structure, these residues were previously shown to exhibit de- adoption of secondary structure, these residues were previously shown to exhibit decreased creased hydrogen–deuterium exchange upon sGC activation [54]. hydrogen–deuterium exchange upon sGC activation [54]. Since there is no direct contact between the regulatory H-NOX/PAS lobe and the catalytic lobe of sGC, structural changes in the CC domains are likely a key driver of allosteric signal transduction. Using their structural observations, Kang et al. engineered single proline mutations into the CC domains of human sGC (α-D423P or β-G356P) to generate a constitutive break in one or the other of the CC helices [65]. Both variants exhibited impaired NO-dependent activation, while the same residues mutated to alanine had no effect on activity [65]. Together, these experiments support the hypothesis that straightening of the CC domains is critical to sGC activation. In addition to straightening, the CC helices also rotate about one another upon activation (Figure8A). When the unactivated and activated structures are superimposed along the α CC domain, the β CC helix undergoes an apparent ~70◦ rotation around the α CC helix (Figure8A). This rotation shifts the C-terminal ends of the CC helices (Figure8B), which is likely important for transmitting signal to the CAT domains, as discussed below. To stabilize the fully active form of moth sGC, Horst et al. included the small molecule sGC stimulator YC-1 in their EM grid preparation [64]. Electron density consistent with the size and shape of YC-1 was observed in the structure between the β H-NOX domain and the α CC domain. Two orientations of YC-1 were possible based on the observed density (Figure8C). When the CC domains of moth sGC are modeled in the correct register, using the structure and sequence of human sGC as a guide, the YC-1 density sits along the stretch of the α CC that is bent in the unactivated enzyme (Figure8C). Although the exact contacts between YC-1 and the protein are unclear given the limiting resolution of

the structure (5.8 Å), the general positioning of YC-1 suggests that it, and related drug

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that are not apparent in the static endpoint snapshots captured by EM. Alternatively, the role of the PAS domains could be mainly structural in facilitating dimerization.

Figure 7. The two sGC PAS domains interact via a conserved dimerization motif and change very little upon activation. Superposition of the unactivated (grey, PDB ID 6JT0 [65]) and activated (colored as in Figure 4, PDB ID 6JT2 [65]) sGC PAS domains. The hydrophobic residues (α-I277, α-F282, α-F286, α-L380, α-L382; β-I208, β-F213, β-F217, β-L320, β-L322) that interact at the dimer interface are shown as sticks. Structures were aligned by secondary structure matching over both chains (r.m.s.d. = 0.96 Å for 234 residues aligned). The N- and C-terminal residues of each domain are labeled and shown as small spheres (C-terminal residue labels have been omitted from the bottom panel).

6.5. The CC Domains The most obvious conformational change that takes place upon sGC activation occurs in the CC domains. In the unactivated structure, each CC domain is divided into two nearly perpendicular helical segments connected by a loop region that straightens upon Int. J. Mol. Sci. 2021, 22, 5439 activation to form two long helices (Figure 8A). These bent loop regions comprise residues13 of 18 α-419–427 and β-355–359. The higher resolution (4.0 Å) structure of the unactivated human sGC allowed for more accurate modeling of the CC register in this region due to the ability to observe electron density for many of the side chains [65]. Consistent with the moleculesadoption likeof secondary Adempas ®structure,[9], may functionthese resi bydues stabilizing were previously the straightened shown conformation to exhibit de- ofcreased the α CC hydrogen–deuterium domain. exchange upon sGC activation [54].

Figure 8. The CC domains play an important role in signal transduction within sGC. (A) Super- position of the CC domains in the unactivated (grey, PDB ID 6JT0 [65]) and activated (colored as in Figure4, PDB ID 6JT2 [ 65]) states. The CC helices straighten upon activation concomitant with rotation of one helix relative to the other, as indicated by black arrows. Structures were aligned by least squares ﬁtting of residues α429–α456 (r.m.s.d. = 1.11 Å for 28 Cα atoms). The N- and C-terminal residues of each domain are labeled and shown as small spheres. (B) View up the length of the CC domains from the C-terminal end. Rotation of the helices leads to a shifting of the C-terminal residues, as indicated by black arrows. (C) Electron density corresponding to YC-1 was observed between the β H-NOX and α CC domains. The two conformations of YC-1 that ﬁt the density are shown in sticks with C in green and cyan, N in blue, and O in red. Residue numbers correspond to the human sGC sequence. β-His105 has been shown in sticks and labeled as a reference point for the αF helix.

6.6. The CAT Domains As predicted from the crystal structures of isolated GC CAT domains, the CAT domains in full-length sGC form a wreath-like heterodimer that undergoes a conformational change upon activation [19–22]. This conformational change involves a clamp-like motion of the β subunit relative to the α subunit (Figure9), as described above. Concomitant with the clamping of the β subunit, the active site opens through the movement of helix α4 of the β CAT domain (residues β-545–555) away from the α CAT domain (Figure9). Kang et al. noted that the net result of these changes is an overall expansion of the central cleft between the two monomers from 1375 to 1549 Å3 [65]. Together, this opening of the central cleft and active site is consistent with the observed decrease in KM(GTP) upon sGC activation [71]. Int. J. Mol. Sci.Int. 2021 J. Mol., 22, x Sci. FOR2021 PEER, 22, REVIEW 5439 14 of 18 14 of 18

Figure 9. The sGCFigure CAT 9. domains The sGC undergo CAT domains a conformational undergo a change conformational upon activation. change Superpositionupon activation. of theSuperposition unactivated (grey, PDB ID 6JT0 [65of]) andthe unactivated activated (colored (grey, PDB as in ID Figure 6JT04 [65]), PDB and ID 6JT2activated [ 65]) (colored sGC CAT as domains.in Figure Clamping4, PDB ID of6JT2 the [65])β subunit sGC CAT domains. Clamping of the β subunit relative to the α subunit is achieved through motions relative to the α subunit is achieved through motions of the β7–β8 loop and the α1 and α2 helices, as indicated by the of the β7–β8 loop and the α1 and α2 helices, as indicated by the curved black arrows. Shifting of the curved black arrows. Shifting of the α4 helix opens the active site for substrate binding, as indicated by the straight black α4 helix opens the active site for substrate binding, as indicated by the straight black arrow. The arrow. The substratesubstrate analog analog GMPCPP GMPCPP of the of activated the activated structure structure is shown is shown as sticks as withsticks C with in green, C in Ngreen, in blue, N in O blue, in red, and P in orange. TheO two in red, magnesium and P in ions orange. are shown The two as greenmagnesium spheres. ions Structures are shown were asaligned green spheres. by least squaresStructures ﬁtting were of the α subunits (r.m.s.d.aligned = 1.13 by Å least for 189 squares Cα atoms). fitting Theof the N- α and subunits C-terminal (r.m.s.d residues. = 1.13 Å of for each 189 domain Cα atoms). are labeled The N- and and shown C- as small spheres. terminal residues of each domain are labeled and shown as small spheres.

The conformationalThe conformational changes in changes the sGC in theCAT sGC domains CAT domains appear appearto be driven to be driven by up- by upstream stream rearrangementsrearrangements within within the theprotein. protein. In particular, In particular, rotation rotation of the of the CC CC helices helices brings brings the two the two C-terminalC-terminal CC CC residues residues into into closer closer proximity (Figure(Figure8 B),8B), thus thus shifting shifting the the N-termini N- of the termini of theCAT CAT domains. domains. This This shift shift then then apparently apparently propagates propagates through through the the rest rest of theof the CAT domain CAT domainarchitecture, architecture, ultimately ultimately resulting resulting in thein the opening opening up up of theof the substrate substrate binding binding site. site. 7. Insight into the 1-NO State from Small Angle X-Ray Scattering 7. Insight into theThe 1-NO cryo-EM State structures from Small presented Angle X-Ray by Horst Scattering et al. [64 ] and Kang et al. [65] represent conformations of sGC that correspond to the two extremes of enzymatic activity—the The cryo-EM structures presented by Horst et al. [64] and Kang et al. [65] represent unactivated state and the fully activated state. sGC, however, exhibits a distinct three-state conformations of sGC that correspond to the two extremes of enzymatic activity—the un- activity proﬁle that includes an intermediary activity state, which is achieved through the activated state and the fully activated state. sGC, however, exhibits a distinct three-state binding of a single equivalent of NO to the sGC heme (Figure1B). This 1-NO state is likely activity profile that includes an intermediary activity state, which is achieved through the to be the physiological resting state, given the estimated intracellular NO concentration binding of a single equivalent of NO to the sGC heme (Figure 1B). This 1-NO state is likely and the Kd of NO release from the heme, as discussed above [37,38]. To provide structural to be the physiologicalinsight into thisresting key state, state ofgiven sGC, the Horst estimated et al. employed intracellular size NO exclusion concentration chromatography and the Kd (SEC)of NO coupledrelease from to small the heme, angle X-rayas discus scatteringsed above (SAXS) [37,38]. to interrogateTo provide solution-statestructural confor- insight intomational this key changesstate of sGC, [64]. TheHorst analysis et al. employed revealed thatsize theexclusion 1-NO statechromatography is best modeled as an (SEC) coupledensemble, to small with angle 72% X-ray of the scattering sample in (SAXS) the unactivated to interrogate conformation solution-state and 28%confor- of the sample mational changesin a partially [64]. The extended analysis conformation. revealed that The the calculated 1-NO state structure is best modeled of this partially as an extended ensemble, withconformation 72% of the lies sample somewhere in the betweenunactivated that conformation of the unactivated and 28% and fullyof the activated sample structures in a partiallyobserved extended by EM.conformation. This apparent The conformationalcalculated structure distribution of this explainspartially theextended partial activity of conformationthe lies 1-NO somewhere state, which between is, on average,that of the ~15% unactivated of the maximum, and fully fully activated activated struc- sGC activity. tures observed by EM. This apparent conformational distribution explains the partial activity of the8. 1-NO Conclusions state, which is, on average, ~15% of the maximum, fully activated sGC activity. The structures reported by Horst et al. and Kang et al. represent a major advance in our understanding of sGC and provide a foundation for elucidating the biochemical 8. Conclusionsdetails of sGC activation. In particular, the rearrangements that occur in the CC domains The structuresupon activation reported appear by Horst to be et critical al. and in Kang communicating et al. represent the a ligation major advance state of the in protein to our understandingthe CAT domainsof sGC and (Figures provide4 and a foun8). Otherdation hints for intoelucidating the signal the transduction biochemical processde- come tails of sGCfrom activation. apparent In contactsparticular, between the rearrangements conserved residues that occur in the in heme-containingthe CC domains β H-NOX upon activationdomain appear and theto beα CCcritical and inβ PAScommu domainsnicating (Figure the ligation6). Together, state theseof the observationsprotein to suggest the CAT domainsa model (Figures for allosteric 4 and communication8). Other hints into in sGC.the signal Namely, transduction shifting of process the β H-NOX come αF helix from apparentresults contacts in loss between of contacts conserved between residues the α inF the helix heme-containing and the CC and β H-NOX PAS domains. do- These main and therearrangements α CC and β PAS promote domains straightening (Figure 6). of Together the CC, domains, these observations which in turn suggest induces a shifts in

Int. J. Mol. Sci. 2021, 22, 5439 15 of 18

the CAT domains that open up the GTP binding site. In this model, the activity states of sGC arise due to shifting of the equilibrium between bent and straight CC helices: basal activity is due to a small, fluctuating population of the unliganded enzyme that is in the elongated conformation. Binding of a first equivalent of NO to the heme cofactor induces higher activity through rupture of the iron–β-His105 bond, favoring the shift of the αF helix. The small molecule stimulator YC-1 shifts this equilibrium further towards high activity by stabilizing the straightened conformation of the α CC helix. Similarly, binding of additional NO to form the excess-NO state would also shift the equilibrium towards straightening of the CC domains, although the mechanism in this case remains unknown. Further mutagenesis studies are needed to provide biochemical support for this model and higher resolution structures of sGC bound to YC-1 would give insight into the contacts involved in stabilizing that interaction. Most importantly, identification of the site, or sites, of excess NO binding will be a critical step towards completing our understanding of sGC activation. Additionally important is developing a better understanding of the 1-NO state of sGC, as this state is likely to be the physiological resting state of the enzyme. The SEC-SAXS analysis by Horst et al. suggested that the 1-NO state contains multiple conformations. Future work to characterize these conformations could include additional advanced SAXS techniques such as ligand titration SAXS coupled to singular value decomposition to identify structural transitions involved in forming the 1-NO state and in transition of the 1-NO state to the excess-NO state [72]. Additionally, performing cryo-EM on the 1-NO state may enable visualization of the different conformations present in the sample. In particular, advances in EM data processing techniques, such as the algorithm cryoDRGN, could facilitate the identification of discrete states and continuous conformational changes in heterogeneous samples [73]. Given these kinds of current, rapid advances in SAXS and EM analyses, there can be no doubt that understanding sGC from a structural perspective is only just beginning.

Author Contributions: Writing—original draft preparation, E.C.W.; writing—review and editing, M.A.M.; visualization, E.C.W.; supervision, M.A.M.; project administration, M.A.M.; funding acquisi- tion, M.A.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by National Institutes of Health (NIH) grant number R01 GM127854. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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